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. 2017 Jun 28;23(24):4354-4368.
doi: 10.3748/wjg.v23.i24.4354.

Naringenin Prevents Experimental Liver Fibrosis by Blocking TGFβ-Smad3 and JNK-Smad3 Pathways

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Free PMC article

Naringenin Prevents Experimental Liver Fibrosis by Blocking TGFβ-Smad3 and JNK-Smad3 Pathways

Erika Hernández-Aquino et al. World J Gastroenterol. .
Free PMC article

Abstract

Aim: To study the molecular mechanisms involved in the hepatoprotective effects of naringenin (NAR) on carbon tetrachloride (CCl4)-induced liver fibrosis.

Methods: Thirty-two male Wistar rats (120-150 g) were randomly divided into four groups: (1) a control group (n = 8) that received 0.7% carboxy methyl-cellulose (NAR vehicle) 1 mL/daily p.o.; (2) a CCl4 group (n = 8) that received 400 mg of CCl4/kg body weight i.p. 3 times a week for 8 wk; (3) a CCl4 + NAR (n = 8) group that received 400 mg of CCl4/kg body weight i.p. 3 times a week for 8 wk and 100 mg of NAR/kg body weight daily for 8 wk p.o.; and (4) an NAR group (n = 8) that received 100 mg of NAR/kg body weight daily for 8 wk p.o. After the experimental period, animals were sacrificed under ketamine and xylazine anesthesia. Liver damage markers such as alanine aminotransferase (ALT), alkaline phosphatase (AP), γ-glutamyl transpeptidase (γ-GTP), reduced glutathione (GSH), glycogen content, lipid peroxidation (LPO) and collagen content were measured. The enzymatic activity of glutathione peroxidase (GPx) was assessed. Liver histopathology was performed utilizing Masson's trichrome and hematoxylin-eosin stains. Zymography assays for MMP-9 and MMP-2 were carried out. Hepatic TGF-β, α-SMA, CTGF, Col-I, MMP-13, NF-κB, IL-1, IL-10, Smad7, Smad3, pSmad3 and pJNK proteins were detected via western blot.

Results: NAR administration prevented increases in ALT, AP, γ-GTP, and GPx enzymatic activity; depletion of GSH and glycogen; and increases in LPO and collagen produced by chronic CCl4 intoxication (P < 0.05). Liver histopathology showed a decrease in collagen deposition when rats received NAR in addition to CCl4. Although zymography assays showed that CCl4 produced an increase in MMP-9 and MMP-2 gelatinase activity; interestingly, NAR administration was associated with normal MMP-9 and MMP-2 activity (P < 0.05). The anti-inflammatory, antinecrotic and antifibrotic effects of NAR may be attributed to its ability to prevent NF-κB activation and the subsequent production of IL-1 and IL-10 (P < 0.05). NAR completely prevented the increase in TGF-β, α-SMA, CTGF, Col-1, and MMP-13 proteins compared with the CCl4-treated group (P < 0.05). NAR prevented Smad3 phosphorylation in the linker region by JNK since this flavonoid blocked this kinase (P < 0.05).

Conclusion: NAR prevents CCl4 induced liver inflammation, necrosis and fibrosis, due to its antioxidant capacity as a free radical inhibitor and by inhibiting the NF-κB, TGF-β-Smad3 and JNK-Smad3 pathways.

Keywords: Carbon tetrachloride; Fibrosis; JNK; Naringenin; Nuclear factor kappa; Smad3; Transforming growth factor-β; pSmad3.

Conflict of interest statement

Conflict-of-interest statement: The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Chemical structure of naringenin.
Figure 2
Figure 2
Naringenin prevents necrosis and cholestasis in CCl4-treated rats. Alanine aminotransferase (ALT) (A); alkaline phosphatase (AP) (B) and γ-glutamyl transpeptidase (γ-GTP) (C) activities were determined in serum from control rats, carbon tetrachloride (CCl4)-treated rats, CCl4 plus naringenin rats (CCl4 + NAR), and rats administered with NAR alone (NAR). Values represent the mean of experiments performed in duplicate assay ± SE (n = 8). aP < 0.05 vs control group; bP < 0.05 vs CCl4 group.
Figure 3
Figure 3
Naringenin prevents glycogen depletion in CCl4-treated rats. Liver glycogen content determined in control rats, carbon tetrachloride (CCl4)-treated rats, CCl4 plus naringenin rats (CCl4 + NAR), and rats administered with NAR alone (NAR). Each bar represents the mean value of experiments performed in duplicate assay ± SE (n = 8). aP < 0.05 vs control group; bP < 0.05 vs CCl4 group.
Figure 4
Figure 4
Naringenin prevents oxidative stress in CCl4-treated rats. Lipid peroxidation (A), reduced glutathione (GSH) determinations in liver (B) and blood (C) and GPx activity (D) from control rats, carbon tetrachloride (CCl4)-treated rats, CCl4 plus naringenin rats (CCl4 + NAR), and rats administered with NAR alone (NAR). Each bar represents the mean value of experiments performed in duplicate assay ± SE (n = 8). aP < 0.05 vs control group; bP < 0.05 vs CCl4 group.
Figure 5
Figure 5
Naringenin prevents inflammation in CCl4-treated rats. The NF-κB (A), IL-1 (B) and IL-10 (C) protein levels in samples of liver tissue were determined by western blot analysis from control rats, carbon tetrachloride (CCl4)-treated rats, CCl4 plus naringenin rats (CCl4 + NAR), and rats administered with NAR alone (NAR). β-actin was used as a control. Values are expressed as fold increase of relative IOD normalized to the control group values (control = 1). Each bar represents the mean value of three rats ± SE. aP < 0.05 vs control group; bP < 0.05 vs CCl4 group.
Figure 6
Figure 6
Naringenin prevents collagen deposition in CCl4-treated rats. Liver collagen content determined in control rats, carbon tetrachloride (CCl4)-treated rats, CCl4 plus naringenin rats (CCl4 + NAR), and rats administered with NAR alone (NAR). Each bar represents the mean value of experiments performed in duplicate assay ± SE (n = 8). aP < 0.05 vs control group; bP < 0.05 vs CCl4 group.
Figure 7
Figure 7
Naringenin effect on macroscopic and microscopic hepatic architecture in CCl4-treated rats. Macroscopic aspect of livers in control rats (A), carbon tetrachloride (CCl4)-treated rats (B), CCl4 plus naringenin rats (CCl4 + NAR) (C), and NAR alone rats (D). Hematoxylin and Eosin stain in livers of control rats (E), CCl4-treated rats (F), CCl4 + NAR rats (G), and rats administered with NAR alone (H). Masson’s trichromic staining in livers of control rats (I), CCl4-treated rats (J), CCl4 + NAR (K), and NAR alone rats (L). Bar scale = 50 μm. Magnification × 100.
Figure 8
Figure 8
Naringenin prevents elevation of metalloproteinase-9 and 2 activities in CCl4-treated rats. Matrix metalloproteinase (MMP)-9 (A) and MMP-2 (B) activities was analyzed by zymography using gelatin-substrate gels (C). Liver samples for control rats, carbon tetrachloride (CCl4)-treated rats, CCl4 plus naringenin rats (CCl4 + NAR), and rats administered with NAR alone (NAR) were analyzed. Values are expressed as average of relative IOD, normalized to the control group values (control = 1). Each bar represents the mean value of three rats ± SE. aP < 0.05 vs control group; bP < 0.05 vs CCl4 group.
Figure 9
Figure 9
Naringenin prevents elevation of TGF-β, α-SMA, CTGF, MMP-13, and Col-1 protein levels, and preserves Smad7 protein levels in CCl4-treated rats. The TGF-β (A), α-SMA (B), CTGF (C), MMP-13 (D), Col-1 (E) and Smad7 (F) protein levels in samples of liver tissue were determined by western blot analysis from control rats, carbon tetrachloride (CCl4)-treated rats, CCl4 plus naringenin rats (CCl4 + NAR), and rats administered with NAR alone (NAR). β-actin was used as a control. Values are expressed as fold increase of relative IOD normalized to the control group values (control = 1). Each bar represents the mean value of three rats ± SE. aP < 0.05 vs control group; bP < 0.05 vs CCl4 group.
Figure 10
Figure 10
Naringenin prevents Smad3 linker phosphorylation by JNK inhibition in CCl4-treated rats. pJNK (A) and pSmad3L protein activation (B) and Smad3 protein levels (C) in samples of liver tissue were determined by western blot analysis from control rats, carbon tetrachloride (CCl4)-treated rats, CCl4 plus naringenin rats (CCl4 + NAR), and rats administered with NAR alone (NAR). β-actin was used as a control. Values are expressed as fold increase of relative IOD normalized to the control group values (control = 1). Each bar represents the mean value of three rats ± SE. aP < 0.05 vs control group; bP < 0.05 vs CCl4 group.
Figure 11
Figure 11
Schematic representation of the antifibrotic effect of naringenin. Naringenin (N) may act at 7 levels to prevent liver fibrosis: (1) by maintaining basal TGF-β levels, thus, preventing hepatic stellate cells activation (HSC), (2) preserving normal metalloproteinases (MMPs) activity blocking TGF-β liberation from extracellular matrix (ECM), (3) increasing the inhibitory protein Smad7, (4) by blocking the activation of JNK, (5) preserving Smad3 levels within control values, (6) blocking the proinflammatory factor NF-κB, (7) by counteracting oxidative stress, preserving reduced glutathione (GSH) levels, glutathione peroxidase (GPx) activity and lipid peroxidation (LPO) induced by reactive oxygen species (ROS), within normal levels.

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